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United States Patent |
5,770,571
|
Cerami
,   et al.
|
June 23, 1998
|
Method and agents for inhibiting protein aging
Abstract
The present invention relates to compositions and methods for inhibiting
the aging of amino-containing amino acid, peptides, proteins and
biomolecules. Accordingly, a composition is disclosed which comprises an
agent or compound capable of reacting with the glycosyl-amino moiety of
the early glycosylation product (also known as the Amadori product or the
Heyns product) formed by the reaction of glucose, or other reactive
sugars, with an amino-containing peptide, protein or biomolecule, thus
stabilizing this early glycosylation product, and preventing its further
reaction to form open-chain, carbonyl-containing advanced glycosylation
end products. Suitable agents may contain a reactive aldehyde group. A
preferred agent is acetaldehyde. The method comprises contacting the
target biomolecule with the composition. Both industrial and therapeutic
applications for the invention are envisioned, as food spoilage and animal
protein aging can be treated.
Inventors:
|
Cerami; Anthony (New York, NY);
Al-Abed; Yousef (New York, NY);
Bucala; Richard J. (New York, NY);
Ulrich; Peter C. (Old Tappan, NJ)
|
Assignee:
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The Picower Institute for Medical Research (Manhasset, NY)
|
Appl. No.:
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746742 |
Filed:
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November 15, 1996 |
Current U.S. Class: |
514/12; 424/130.1; 426/133; 426/321; 514/2; 524/12; 530/300; 530/350 |
Intern'l Class: |
A61K 038/00; C08J 089/06; C07K 013/00; A21D 004/00 |
Field of Search: |
524/21
424/130.1
514/2,12
530/300,350
426/133,321
|
References Cited
U.S. Patent Documents
4665192 | May., 1987 | Cerami et al.
| |
Other References
Hayase et al., J. Biol. Chem., 263, pp. 3758-3764 (1989).
Sell et al., "Structure Elucidation of a Senescence Cross-link from Human
Extracellular Matrix", J. Biol. Chem., 264, pp. 21597-21602 (1989).
|
Primary Examiner: Jones; W. Gary
Assistant Examiner: Rees; Dianne
Attorney, Agent or Firm: Klauber & Jackson
Claims
What is claimed is:
1. A compound of the formula VI or VIII
##STR6##
wherein R is the residue of an amino-containing amino acid, peptide,
protein, biomolecule or compound,
said compound of the formula VI or VIII prepared by reaction of the
glycosyl-amino moiety of the early glycosylation product (also known as
the Amadori product or the Heyns product) formed by the reaction of
glucose, or other reactive sugars, with said amino-containing amino acid,
peptide, protein, biomolecule, or compound with an agent containing a
reactive aldehyde group; and
CH.sub.2 R' the residue of the reactive aldehyde group wherein R' is an
alkyl group of 1-12 carbons, an alkenyl group of 1-20 carbon atoms
containing 1-4 degrees of unsaturation, and alkynyl group of 1-20 carbon
atoms containing one or more triple bonds, an aryl or a heteroaryl group,
each of which can optionally be substituted by one or more halogen or
hydroxyl groups.
2. The compound according to claim 1, wherein said agent containing a
reactive aldehyde group is acetaldehyde, or an agent which is converted
metabolically in vivo to acetaldehyde.
3. The compound according to claim 1, which is
1-deoxy-1-alkylamino-1,2-N,O-ethylidene-.beta.-D-fructopyranose.
4. The compound according to claim 1, which is 1-deoxy-1-alkylamino-1,3-N,
O-ethylidene-.beta.-D-fructopyranose.
Description
This Application claims priority from United States Provisional Application
60/006,752, filed Nov. 15, 1995.
This invention was made with partial assistance from a grant, DK19655
"Biochemical Basis of the Complications of Diabetes," from the National
Institute of Diabetes and Digestive and Kidney Diseases of the National
Institutes of Health.
BACKGROUND OF THE INVENTION
The present invention relates generally to the aging of proteins and other
biomolecules resulting from reaction of glucose, and particularly to the
non-enzymatic glycation or glycosylation of proteins and other susceptible
amine-presenting molecules and subsequent reactions leading to advanced
glycosylation end products, and to methods and agents for their
inhibition.
The reaction between glucose and proteins has been known for many years.
Its earliest manifestation was in the appearance of brown pigments during
the cooking of food, which was identified by Maillard in 1912, who
observed that glucose or other reducing sugars react with amino-containing
compounds, including amino acids and peptides, to form adducts that
undergo a series of dehydrations and rearrangements to form stable brown
pigments.
In the years that followed the initial discovery by Maillard, food chemists
studied this reaction in detail and determined that stored and
heat-treated foods undergo non-enzymatic browning as a result of the
reaction between glucose and the polypeptide chain, and that the proteins
are resultingly crosslinked and correspondingly exhibit decreased
bioavailability. At this point, it was determined that the pigments
responsible for the development of the brown color that develops as a
result of protein glycosylation possessed characteristic spectra and
fluorescent properties.
The reaction between reducing sugars and food constituents discussed above
was found in recent years to have its parallel in vivo. Thus, the
non-enzymatic reaction between aldehyde sugars such as glucose, galactose
and arabinose and the free amino groups on proteins to form a stable
amino, 1-deoxy ketosyl adduct, is known as the Amadori product. In the
case of ketone sugars such as fructose, this non-enzymatic reaction
product is known as the Heyns product, with reactivities parallel to that
of an Amadori product. This reaction has been shown to occur with
hemoglobin, wherein a rearrangement of the amino terminus of the
.beta.-chain of hemoglobin, following an initial reaction with glucose,
forms the modified hemoglobin known as hemoglobin A.sub.1c. Similar
reactions have also been found to occur with a variety of other peptides,
proteins, both soluble and structural, and biomolecules, such as lens
crystallins, collagen nerve proteins, and low density lipoproteins, DNA
and aminophospholipids.
As a result of the recent interest in this area, the first few stages of
the Maillard reaction, and a relatively limited number of associated
initial adducts and products, have become well-known. As subsequent
reactions (including various dehydrations, oxidations, eliminations,
condensations, cleavages, and other chemical changes) occur, however, a
bewildering array of "early" and "late" glycation adducts and reactants is
generated, and these are less well understood in molecular detail. As a
group, the more advanced glycation adducts can be described as a class of
yellow-brown, fluorescent pigments with intra- and intermolecular
crosslinking activity, wherein specific glycation entities are thought to
occur at low abundance within the widely divergent pool of advanced
glycation end products (or AGEs). Despite significant work over the last
twenty years or so, the molecular structures of only a few of these later
glycation adducts and products have been determined, and the contribution
of identified, in vivo-formed advanced glycation structures to specific
biological processes remains poorly understood.
In U.S. Pat. No. 4,665,192 the fluorescent chromophore
2-(2-furoyl)-4(5)-2(furanyl)-1H-imidazole was isolated and identified from
certain browned polypeptides such as bovine serum albumin and
poly-L-lysine. This chromophore made possible the identification of the
advanced glycosylation end products and assisted additional investigations
seeking to clarify the protein aging process and to identify the specific
chemistry involved in order to develop methods and agents for its
inhibition.
More recently, other advanced glycation products have been identified, such
as Farmar et al., U.S. Ser. No. 097,856, filed Sep. 17, 1987; pyrraline
(Hayase et al., "Aging of Proteins: Immunological Detection of a
Glucose-derived Pyrrole Formed during Maillard Reaction in Vivo", J. Biol.
Chem., 263, pp. 3758-3764 (1989)); and pentosidine (Sell et al.,
"Structure Elucidation of a Senescence Cross-link from Human Extracellular
Matrix", J. Biol. Chem., 264, 15 pp. 21597-21602 (1989)).
A large body of evidence has been assembled to show that Maillard products
as a whole underlie a wide variety of both normal and pathogenic
activities and responses that occur as advanced glycation end products (or
AGEs) accumulate in vivo. Such activity may be direct, as a consequence of
the chemical reactivity of glycation products and adducts, or indirect,
mediated by the cellular recognition of glycation adducts and products via
AGE-specific binding proteins or receptors. An appreciation for the
pathogenic potential of AGEs has suggested that interference with, or
inhibition of, advanced glycation chemistry could be of enormous
therapeutic benefit. The agent pimagedine (aminoguanidine), and other
related compounds, have been found to be useful glycation inhibitors. This
compound, and others like it, has been theorized to react with the
carbonyl moiety of the early glycosylation product of a target protein
formed subsequent to the initial non-enzymatic reaction with glucose or
another reducing sugar, and thereby prevent further reaction to form
open-carbonyl-containing advanced glycosylation end products.
Although pimagedine has shown a great therapeutic potential, there exist a
need to discover and develop alternative glycation inhibitors, active, for
instance, at different stages of the Maillard reaction and/or against a
different spectrum of glycation intermediates and AGEs. Such alternates
would provide additional treatment modalities against the deleterious
sequelae of AGE accumulation in vitro and in vivo. The present invention
is thus directed toward inhibition of the Maillard reaction, and is shown
to operate through a mechanism not exploited previously in this regard.
Recently, it has been discovered that other naturally-occurring reducing
sugars, including fructose, ribose and galactose, participate in
non-enzymatic glycation and cross-linking. Because the methods and agents
of the present invention block non-enzymatic crosslinking mediated by any
such reactive sugars, they are expected to prevent fructose-mediated
crosslinking as well. Cross-linking caused by other reactive sugars
present in vivo or in foodstuffs, including ribose and galactose, would
also be prevented by the methods and compositions of the present
invention.
SUMMARY OF THE INVENTION
In accordance with the present invention, an improved method and associated
agents are disclosed for the inhibition of the aging of amino-containing
amino acids, peptides, proteins and biomolecules. In particular, agents
for inhibiting protein aging due to the formation of advanced
glycosylation end products may be selected from those materials capable of
reacting with the glycosyl-amino moiety of the early glycosylation product
(also known as the Amadori product or the Heyns product) formed by the
reaction of glucose, or other reactive sugars, with an amino-containing
peptide, protein, or biomolecule, thus stabilizing this early
glycosylation product, and preventing its further reaction to form
undesired open-chain, carbonyl-containing advanced glycosylation end
products. Thus, for example, compounds or compositions having an active
aldehyde substituent are suitable, and especially, compounds such as
acetaldehyde. The molecular basis of action of these agents appears to
involve the reaction with the early glycosylation product formed between
glucose, or other reactive sugars, and an amino-containing peptide,
protein, or biomolecule whereby the resulting tripartite reaction product
is stable, precludes rearrangement of the early glycation product into an
open-chain, carbonyl-containing configuration, and thus does not support
further reactions that would continue the advanced glycation process.
Irrespective of their underlying mechanism of action, these agents prevent
the formation of advanced glycosylation end products, and associated
molecular changes, such as crosslinks.
The present invention also relates to a method for inhibiting aging of
amino-containing peptides, proteins or biomoleules by contacting the
initially glycosylated protein at the stage of the early glycosylation
product with a quantity of one or more of the agents of the present
invention. In the several instances where the present method has
industrial application, one or more of the agents may be applied to the
peptides, proteins or biomolecules in question, either by introduction
into a mixture of the same in the instance of a protein extract, or by
application or introduction into foodstuffs containing the protein or
proteins, all to prevent premature aging and spoilage of the particular
foodstuffs and other comestibles. Other industrial amine-containing
compounds and products, including, for instance, various drugs, various
nutritional ingredients for parenteral administration and the like, may
likewise benefit from treatment with agents of and/or by the methods of
the present invention. The agents and methods of the present invention can
be used to extend the useful storage life of such amino-group containing
commercial products, by inhibiting the formulation of AGEs on said amino
groups during storage of said product.
In the instance where the present method has therapeutic application, the
animal host intended for treatment may have administered to it a quantity
of one or more of the agents, in a suitable pharmaceutical form.
Administration may be accomplished by known techniques, such as oral,
topical and parenteral techniques such as intradermal, subcutaneous,
intravenous, or intraperitoneal injection, as well as by other
conventional means such as inhaled aerosols or nemubulized droplets.
Administration of the agents may take place over an extended period of
time at a dosage level of, for example, up to about 25 mg/kg.
The ability to inhibit the formation of advanced glycosylation end products
carries with it significant implications in all applications where protein
aging is a serious detriment. Thus, in the area of food technology, the
retardation of food spoilage would confer an obvious economic and social
benefit by making certain foods of marginal stability less perishable and
therefore more available for consumers. Spoilage would be reduced, as
would the expense of inspection, removal and replacement, and the extended
availability of the foods could aid in stabilizing their price in the
marketplace. Similarly, in other industrial applications where the
perishability of proteins or other amino-containing biomolecules (e.g.
lipids and DNA) or compounds (e.g. pharmaceutical compositions) is a
problem, the admixture of the agents of the present invention in
compositions containing such peptides, proteins, or biomolecules would
facilitate the extended useful life of the same. Presently used food
preservatives and discoloration preventatives such as sulfur dioxide,
known to cause toxicity including allergy and asthma in animals, might be
replaced with compounds such as those described herein.
The present method has particular therapeutic application as the Maillard
process acutely affects several of the significant protein masses in the
body, among them collagen, elastin, lens proteins, and the kidney
glomerular basement membranes. These proteins deteriorate both with age
(hence the application of the term "protein aging") and as one of the
sequelae of diabetes melitus. Consequently, the ability to either retard
or substantially inhibit the formation of advanced glycosylation end
products carries the promise of favorably treating significant adverse
effects of aging and of diabetes and, of course, improving the quality and
perhaps duration of animal life, including for instance human life.
Accordingly, it is a principal object of the present invention to provide a
method for inhibiting the extensive cross-linking of amino-containing
peptides, proteins, biomolecules or other compounds that occurs as an
ultimate consequence of the reaction of said peptides, proteins,
biomolecules or other compounds with glucose or other reducing sugars, by
inhibiting the corresponding formation of advanced glycosylation end
products.
It is a further object of the present invention to provide a method as
aforesaid which is characterized by a reaction with early glycosylation
products.
It is a further object of the present invention to provide a method as
aforesaid which prevents the rearrangement, cross-linking and other
Maillard reactions of the said early glycosylation products to form the
said advanced glycosylation end products.
It is a yet further object of the present invention to provide agents
capable of participating in the reaction with the said early glycosylation
products in the method as aforesaid.
It is a still further object of the present invention to provide
therapeutic methods of treating the adverse consequences of aging,
manifest, for instance, in the stiffening and embrittlement of animal
protein and the browning and spoilage of foodstuffs and other comestibles.
Other objects and advantages will become apparent to those skilled in the
art from a consideration of the ensuing description which proceeds with
reference to the following illustrative drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a comparison of aliquots from incubations of CBZ-lysine-AP, with
and without acetaldehyde, analyzed for immunoreactive AGE epitopes using
an ELISA procedure based on a polyclonal anti-AGE antibody preparation.
FIG. 1B is a comparison graph of aliquots from incubations of
CBZ-lysine-AP, with and without acetaldehyde, at various time points
during the incubation period, analyzed for immunoreactive AGE epitopes
using an ELISA procedure based on a polyclonal anti-AGE antibody
preparation.
FIG. 2 is a comparison of aliquots from incubations of CBZ-lysine-AP, with
and without acetaldehyde, analyzed for immunoreactive AGE epitopes using
an ELISA procedure based on a monoclonal anti-AGE antibody preparation.
FIG. 3 is an HPLC analysis of the incubation mixture of CBZ-lysine-AP with
acetaldehyde.
FIG. 4 is a partial upfield NMR spectrum of an HPLC-purified. fraction of
the incubation mixture of CBZ-lysine-AP with acetaldehyde.
FIG. 5 is a mass spectrum of an HPLC-purified fraction of the incubation
mixture of CBZ-lysine-AP with acetaldehyde.
FIG. 6 is a bar graph comparing the Hb-AGE values determined from blood
samples taken at 8 weeks after the initiation of the study.
FIG. 7 are plots of HPLC (ion exchange chromotography) profiles of the
hemolysates obtained from four groups of rats displayed in panels (a-d).
FIG. 8 shows the plots of a reverse phase liquid chromatography connected
to an electrospray ionization mass spectrometer (LC-MS) of fractions
containing Hb.sub.0 and HbA.sub.1c -acetadehyde adduct from the hemolysate
of alcohol-treated diabetic rats.
FIG. 9A is the ESMS spectra of the .beta.-chain of the HbA.sub.1c
-acetaldehyde adduct.
FIG. 9B is the ESMS spectra of Hb.sub.0.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, compositions and associated
methods have been developed which inhibit the formation of advanced
glycosylation end products in a number of amino-containing amino acids,
peptides, proteins and biomolecules existing in both animals and plant
material. In particular, the invention relates to compositions which may
contain one or more agents for inhibiting aging due to the formation of
advanced glycosylation end products, said agents selected from those
materials capable of reacting with the glycosyl-amino moiety of the early
glycosylation product (also known as the Amadori product or the Heyns
product) formed by the reaction of glucose, or other reactive sugars, with
a molecule bearing one or more amino groups, such as an amino acid,
peptide, protein, other biomolecule or other organic compound, thus
stabilizing said early glycosylation product, and preventing its further
reaction to form open-chain, carbonyl-containing advanced glycosylation
end products.
Reaction Scheme I, shown below, illustrates the first steps of the Maillard
reaction, which describes the sequential spontaneous reactions of reducing
sugars, with glucose being illustrated in the scheme, with
amino-containing compounds, such as the .epsilon.-amino group of a lysine
residue in a peptide or protein. The Maillard reaction thus begins with
the non-enzymatic and readily reversible reaction between a sugar molecule
(Ia or Ib) and an amino group of a peptide or protein (II) to form a
Schiff base (IIa or IIb), which can then rearrange to form the Amadori
product (IVa or IVb). Although illustrated by a series of open chain
formulae (Ia, IIIa and IVa), it should be understood that the open form of
each of these carbohydrate-protein adducts is in equilibrium with the
corresponding closed ring form (Ib, IIIb and IVb), and this closed ring
form generally predominates. More than 99% of the Amadori compound of
formula IV is thus present in the closed ring configuration, which may
involve either an .alpha. or .beta. ring closure at carbon 2 (C2). The
open chain Amadori product of formula IVa is free to dehydrate to generate
the early glycation product 1-alkylamino-1,4-dideoxyglucosone (IX). These
subsequent glycation products, especially the typical diketone type of
formula IX, are believed to represent the class of substrates that can
react with the advanced glycation inhibitor pimagedine (aminoguanidine).
##STR1##
In Scheme I, the remaining portion of the amino acid, peptide, protein or
other molecule bearing the amino substituent is denoted by R. Acetaldehyde
is shown as the agent reactive with the glycosyl-amino moiety of the early
glycosylation product of the amino acid, peptide, protein or other
molecule bearing the amino substituent but other aldehydes may be
substituted as descibed herein. Reactive sugars other than glucose will
produce the corresponding Amadori product (e.g. the aldoses arabinose or
galactose) or a Heyns product (e.g. the ketose fructose).
Applicants have now discovered that, as a distinct alternative approach to
the inhibition the formation of advanced glycosylation end products (AGEs)
by aminoguanidine-type inhibitory agents which target reactive carbonyls
to inhibit more advanced stages of the Maillard reaction, a novel class of
inhibitors can be utilized at an earlier stage, the typical Amadori
product of Formula IV. Specifically, the glycosyl-aminoalkyl moiety of the
Amadori product of formula IV can be reacted with an agent containing a
reactive aldehyde group (illustrated in Scheme I by acetaldehyde, a
representative species of the present invention) to form the unisolated
intermediates of formulae V or VII, which then rearrange to form the
stabilized closed configuration adducts of formulae VI and VIII,
respectively. These adducts of formulae VI and VIII are stable, closed
ring structures which are blocked from participating in subsequent steps
of the advanced glycation (or Maillard) reaction, thus inhibiting the
browning and crosslinking associated with AGE formation.
The thus-formed adducts of formulae VI and VIII thus eliminate the
possibility of further typical glycation reactions, and the detrimental
consequences which flow therefrom and result in the development in vivo of
conditions such as skin wrinkling, certain kidney diseases,
atherosclerosis, osteoarthritis and the like. Similarly, amino-containing
plant, animal and chemical materials that undergo non-enzymatic browning
deteriorate and, in the case of foodstuffs or other comestibles, become
spoiled or toughened and consequently, unpalatable or inedible. Thus, the
reaction of the compounds of the present invention with a susceptible
early stage glyco-amino group is believed to inhibit the late stage
Maillard effects and intervene in the deleterious changes described above.
The rationale of the invention is to use agents which covalently lock early
stage glycation adducts into unreactive tripartite products, thus blocking
later post-glycosylation steps, e.g., the formation of fluorescent
chromophores whose presence is associated with, and leads to, the adverse
sequelae of diabetes and aging. An ideal agent would prevent the formation
of advanced AGE chromophores and associated or independent glycation
cross-links, which can interconnect protein domains intra- or
intermolecularly and covalently trap soluble proteins onto matrix
proteins, as occurs in arteries and in the kidney. This crosslinking may
itself be undesirable, as may be the cellular responses to crosslinked or
other AGE-modified tissue and circulating components.
The present invention does not attempt to prevent the most initial steps of
protein glycosylation, as the reaction of glucose with protein amino
groups is very rapidly reversible and shortlived. Instead, the agents of
the present invention are directed to the Amadori and Heyns rearrangement
products which are less rapidly reversible and accumulate to a significant
degree. Covalent interaction to form the addition products of the present
invention serves to prevent or inhibit the long-term, late glycosylation
steps that lead to the formation of advanced glycosylation end products
that are a direct cause of the pathology associated with aging and
diabetes.
It is the amine group of a protein or other biomolecule which initially
reacts with the glucose, or another reactive sugar, to form an Amadori or
Heyns rearrangement product, which is believed to react with the compounds
of the present invention to form a terminal tripartite addition product.
Accordingly, the compositions useful in the present invention comprise or
contain one or more agents for inhibiting protein aging due to the
formation of advanced glycosylation end products, and may be selected from
those materials capable of reacting with the early glycosylation product
(also known as the Amadori product or the Heyns product) formed from the
reaction of glucose, or other reactive sugars, with a free amino group on
a second molecule such as a protein, thus stabilizing this early
glycosylation product, and preventing its further reaction to form
open-carbonyl-containing advanced glycosylation end products.
Suitable agents include compounds having a reactive aldehyde group which
are represented by the general formula
##STR2##
wherein R' is an alkyl group of 1-12 carbons, an alkenyl group of 1-20
carbon atoms containing 1-4 degrees of unsaturation, and alkynyl group of
1-20 carbon atoms containing one or more triple bonds, an aryl or a
heteroaryl group, each of which can optionally be substituted by one or
more halogen or hydroxyl groups. Also utilizable in the present invention
are compounds which contain group which is metabolically transformed into
an reactive aldehyde group.
For example, the agent may comprise an aldehyde compound such as
acetaldehyde, or an alcohol, such as ethanol, which is metabolized in vivo
to a corresponding aldehyde, in this case, acetaldehyde. Reaction of the
agents of the present invention with the glycosyl-amino moiety, e.g. of a
glycation-modified protein, would lock this nitrogen into a stable ring
system and thus prevent an early glycation product from progressing to
form undesired products, such as crosslinks with other groups.
Thus, the agents of the present invention have been identified and tested
on the basis of their ability to react with the glycosyl-amino moiety of
the Amadori or Heyns rearrangement glycosylation product to form a highly
stable class of adducts. These agents contain a reactive aldehyde group,
or a functional group which is metabolized in vivo to a reactive aldehyde
group.
A representative agent of the present invention comprises acetaldehyde.
This compound is known to have low toxicity in animals. According to the
Handbook of Toxicology, Vol. 1, acetaldehyde base has a LD.sub.50 when
administered orally of 1.9 g/kg in rats. It can thus be utilized to react
with the glycosyl-amino moiety of the Amadori products of formula IV to
form the stable, non-reactive adducts of formula VI and VIII.
Other agents which have utility in the compositions and methods of the
present invention are compounds which contain reactive aldehyde groups
such as:
Phenylacetaldehyde;
2-Phenylpropionaldehyde;
Cinnamaldehyde;
.alpha.-Methylcinnamaldehyde;
.alpha.-Hexylcinnamaldehyde;
o-Methoxycinnamaldehyde;
Benzaldehyde;
o-Anisaldehyde;
Salicylaldehyde;
4-Ethylbenzaldehyde;
Cuminaldehyde;
p-Anisaldehyde;
4-Hydroxybenzaldehyde;
p-Ethoxybenzaldehyde;
2,4-Dimethylbenzaldehyde;
.beta.-Cyclocitral;
3,4-Dihydroxybenzaldehyde;
Veratraldehyde;
Piperonal;
Vanillin;
Ethyl Vanillin;
Acetaldehyde;
Propionaldehyde;
Isobutyraldehyde;
Butyraldehyde;
2-Methybutyraldehyde;
2-Ethybutryaldehyde;
3-Methybutyraldehyde;
Valeraldehyde;
2-Methylpentenal;
Hexanal;
Heptanal;
Octanal;
Nonanal;
Decanal;
Laurio aldehyde;
3-(Methylthio)butanal;
Pyruvaldehyde;
trans-2-Pentenal;
trans-2-Methyl-2-butenal;
3-Methyl-2-butenal;
trans-2-Hexenal;
trans-2-Heptenal;
cis-4-Heptenal;
2,6-Dimethyl-5-heptenal;
trans-2-Octenal;
2-Isopropyl-5-methyl-2-hexenal;
2-Nonenal;
cis-6-Nonenal;
2-Decenal;
10-Undecenal;
trans,trans-2,4-Octadienal;
trans,trans-2,4-Nonadienal;
trans-2,cis-6-Nonadienal;
trans,trans-2,6-Nonadienal;
2,4-Hexadienal;
trans,trans-2,4-Decadienal;
(S)-(-)-Perillaldehyde; and
(1R)-(-)-Myrtenal.
The use of such compounds would result in compounds of formula VI and VIII
wherein the methyl group of acetaldehyde, as illustrated, is replaced by
the non-reactive portion of a compound containing the active aldehyde
group.
As herein noted, also equivalent to the agents of the present invention
containing a reactive aldehyde group are those agents containing a
functional group which is metabolically converted in vivo to a reactive
aldehyde group. For instance, ethyl alcohol or ethanol, when metabolized
by the body, produces acetaldehyde as the principal product. Ethyl alcohol
can thus be used as a therapeutic agent for in vivo applications of the
present invention.
The measurement of the adducts of formula VI and VIII present in an animal
or other protein-containing material can provide an indication of the
amount of reactive aldehyde to which the animal or other
protein-containing material has been exposed, thus leading to both
diagnostic and monitoring utilities for this invention.
For instance, since ethyl alcohol produces acetaldehyde in vivo, the
measurement of the corresponding adducts of formula VI and VIII formed
from acetaldehyde can likewise be used to assess or monitor the ingestion
of ethyl alcohol over periods of time. The present invention can thus be
utilized to make an assessment of the alcohol ingestion by a patient over
a period of time, since it would be expected that these adducts of formula
VI and VIII will be stable and accumulate over time, and be identifiable
in the patient's sera or other body fluids. The present invention thus
finds utility in the area of screening for individuals who have, over a
period of time, ingested ethyl alcohol, thus accumulating detectable
amounts of the adducts of formula VI and/or VIII as a result of alcohol
intake, and serving as a convenient marker of extended alchohol use or
abuse. Similarly, the present invention finds utility in monitoring
individuals for exposure to environmental, inhaled or ingested aldehydes,
such as formaldehyde, and finds further utility in the preparation of
dosimeter-type assays for the integrated exposure to reactive aldehydes.
The findings of the present invention can also be utilized to screen for
additional agents which would have utility as agents for inhibiting
advanced glycation. Thus, the measurement of the amount of the formation
of the adducts of formula VI and/or VIII wherein the methyl group of the
reacted acetaldyde, as illustrated, is replaced by the bulk of the test
aqent bearing the reactive aldehyde of interest, would enable one to
assess the usefulness of an agent as a potential inhibitor of the advanced
glycation process.
The adducts of formula VI and VIII, or variants thereof comprising
different aldehyde-bearing or amino-bearing reactants, may be used in
standard fashion to prepare either polyclonal or monoclonal antibodies
thereto for diagnostic purposes. Such antibodies are preparable by
standard procedures, and thus enable the use of diagnostic assays for
assessing and monitoring the effectiveness of therapeutic regimens where
AGE inhibition has been initiated. Said immunological regents directed
against generic and specific structures of the present invention are also
useful to detect the degree of modification of Amadori or Heyns
rearrangement products in a sample from a subject animal, including, for
example, a human being, thereby to infer the recent history of alcohol
consumption by said subject, by reference to a standard. Said polyclonal
or monoclonal immunological reagents can optionally be included in a kit,
with instructions, and, optionally, a standardized preparation of a
tripartite compound of the present invention, to facilitate such
determinations all as contemplated hereunder.
In the instance where the composition of the present invention is utilized
for in vivo or therapeutic purposes, it may be noted that the compounds or
agents used therein are biocompatible. Pharmaceutical compositions may be
prepared with a pharmaceutically effective quantity of the agents or
compounds of the present invention and may include a pharmaceutically
acceptable carrier, selected from known materials utilized for this
purpose. Such compositions may be prepared in a variety of forms,
depending on the method of administration. For example, a liquid form
would be utilized in the instance where administration is by intravenous
or intraperitoneal injection, which liquid might be aerosolized for
delivery by inhalation; while, if appropriate, tablets, capsules, etc.,
may be prepared for oral administration. For application to the skin, a
lotion or ointment may be formulated with the agent in a suitable vehicle,
perhaps including a carrier to aid in penetration into the skin. Other
suitable forms for administration to other body tissues are also
contemplated.
The present invention likewise relates to methods for inhibiting the
formation of advanced glycosylation end products, which comprise
contacting the target proteins with a composition of the present
invention. In the instance where the target proteins are contained in
foodstuffs or other comestibles, whether of plant or animal origin, these
foodstuffs could have applied to them by various conventional means a
composition containing the present agents. Similarly, amino-containing
compounds such as drugs or ailimentary supplements that deteriorate or
otherwise become spoiled over time by advanced glycation can be protected
by treatment with an agent of the present invention that is biocompatibly
non-toxic, and does not inactivate the desired characteristics of the
compound in need of preservation. Protocols are provided for the
identification of agents of the present invention useful in such contexts.
Likewise, in the instance where therapeutic applications are intended, the
animals to be treated would have administered to them a regular quantity
of the pharmaceutical composition of the present invention. Administration
could take place, for example, daily, and an effective quantity of the
agent or compound of the present invention could range up to 25 mg/kg of
body weight of the animal. A topical preparation may, for example, include
up to 10% of the agent or composition in an ointment or lotion for
application to the skin. Naturally, some variation in these amounts is
possible, and the suggested amounts are provided in fulfillment of
applicants' duty to disclose the best mode for the practice of the present
invention.
As is apparent from a discussion of the environment of the present
invention, the present methods and compositions hold the promise for
arresting the aging of key molecules, whether in animal or plant material,
or in chemical preparations, and whether in vivo or in vitro, and
concomitantly conferring both economic and medical benefits as a result
thereof. In the instance of in vitro use for preserving consumable
materials, such as foodstuffs and other comestibles, from undersired
deterioration, the administration of the present compositions holds the
promise of retarding spoilage and thereby increasing shelf life and
providing more convenience and greater availability to consumers.
Replacement of currently-used preservatives, such as sulfur dioxide known
to cause allergies and asthma in humans, with non-toxic, biocompatible
compounds is a further advantage of the present invention.
The in vivo therapeutic implications of the present invention relate to the
arrest of several of the pathogenic activities associated with the aging
process which have, as indicated earlier, been identified in the aging of
key tissue and circulating proteins by advanced glycosylation and
crosslinking. Thus, body proteins, and particularly structural body
proteins such as collagen, elastin, lens proteins, nerve proteins and
kidney glomerular basement membranes would all benefit in their longevity
and operation from the practice of the present invention. The present
invention thus reduces the senescence caused by pathologies involving the
entrapment of proteins by crosslinked target proteins, as exemplified, for
instance, in retinopathy, cataracts, diabetic kidney disease,
glomerulosclerosis, peripheral vascular disease, arteriosclerosis
obliterans, peripheral neuropathy, stroke, hypertension, atherosclerosis,
osteoarthritis, periarticular rigidity, loss of elasticity and wrinkling
of skin, stiffening of joints, glomerulonephritis, etc. Likewise, all of
these conditions are in evidence in patients afflicted with diabetes
mellitus. Thus, the present therapeutic method is relevant to treatment of
the noted conditions in patients either of advanced age or those suffering
from one of the mentioned pathologies, particularly in association with
hyperglycemia, which accelerates glycation-mediated senescence.
Protein crosslinking through advanced glycosylation product formation can
decrease solubility of structural proteins such as collagen in vessel
walls, and as well as trap serum proteins, such as lipoproteins to the
collagen. Also, this may result in covalent trapping of extravasated
plasma proteins and lipoproteins in subendothelial matrix, and reduction
in susceptibility of both plasma and matrix proteins to physiological
degradation by enzymes. For these reasons, the progressive occlusion of
diabetic vessels induced by chronic hyperglycemia has been hypothesized to
result in part from excessive formation of glucose-derived adducts and
crosslinks. Such diabetic macrovascular changes and microvascular
occlusion can be effectively prevented by chemical inhibition of advanced
glycosylation product formation utilizing a composition and the methods of
the present invention.
Taken together, these data strongly suggest that inhibition of the
formation of advanced glycosylation end products (AGEs), by the teachings
of the present invention, may prevent late as well as early structural
lesions due to diabetes, as well as changes during aging caused by the
formation of AGEs.
The present invention will be better understood from a consideration of the
following illustrative examples, reviewing the selection and testing of
certain of the agents of the present invention on both an in vitro and in
vivo basis.
This invention may be embodied in other forms or carried out in other ways
without departing from the spirit or essential characteristics thereof.
The present disclosure is therefor to be considered as in all respects
illustrative and not restrictive, the scope of the invention being
indicated by the appended claims, and all changes which come within the
meaning and range of equivalency are intended to be embraced therein.
Specifically, Applicants describe herein the covalent reaction of aldehyde
agents with the Maillard Amadori product (or equivalent Heyns
rearrangement product) (illustrative intermediates shown in Scheme I as V
and VII) to consequently form a tripartite product, characterized by the
presence of two adajacent ring systems, said rings being either spiro or
fused bicylic, wherein said tripartite product (e.g., VI and VIII) is
stabilized against progression in the typical reactions of advanced
glycation. Applicants show that this structural "locking" of the Amadori
(or Heyns) structure in a closed configuration prevents the typical
subsequent glycation reactions and inhibits the browning and crosslinking
associated with AGE formation. The reaction mechanisms implied by the
illustrative structural schemes are for explanatory purposes only, and
should not be taken as a limitation on the scope of the present invention.
EXAMPLE 1
Structural confirmation of the Amadori-acetaldehyde adduct (AAA)
A simplified test reaction system was set up in vitro to provide evidence
that aldehydes can react usefully with Amadori products, thus trapping
these glycation intermediates in a closed and unreactive conformation and
thereby largely prevent the process of advanced glycation. As shown in
Scheme II below, a modified, lysine-based Amadori product was synthesized
as a controlled starting material for advanced glycation reactions. This
reagent, CBZ-lysine-AP, was prepared to be susceptible to advanced
glycation at only one location (the .alpha.-amino function of lysine was
blocked by a carbobenzoxy group, leaving only the .epsilon.-amino group
available for glycation reactions). This model Amadori product was then
incubated in the presence or absence of acetaldehyde, as a model agent of
the present invention, both to test the effect of the supplied aldehyde
agent on the progression of advanced glycation as well as to provide
material for structural characterization to confirm the spontaneous
formation of the expected Amadori-acetaldehyde adduct (AAA).
##STR3##
Preparation of N-(1-deoxy-D-fructos-1-yl)-N.sup.a
-carboxybenzoyloxy-lysine or CBZ-lysine-AP
A suspension of 3.6 g (0.02 mol) of anhydrous D-glucose and 0.2 g of sodium
bisulfite in 6 ml of methanol and 3 ml of glycerol was refluxed for 30
min, followed by addition of 7 mmol of N.sup.a -carboxybenzoyloxy-lysine
and 0.8 ml of acetic acid. This solution was refluxed until most of the
starting material disappeared as evidenced by thin layer chromatography
(TLC). TLC was performed on Silica Gel-60 manufacturer glass plates using
the following irrigant (v/v): 4:1:1 n-butanol/acetic acid/water. Plates
were sprayed with 0.2% ninhydrin in ethanol (for detection of lysine and
sugar), followed by heating at 100.degree. C. for about 5 minutes. The
Amadori product was dissolved in methanol and poured into n-propanol with
vigorous stirring. After standing for about 3 hours at room temperature, a
yellowish oily material came out of solution. This upper layer was
decanted and part of the oily material was subjected to preparative high
performance liquid chromatography (HPLC) as follows. A Primesphere
(Phenomenex, Torrance, Calif.) 5 m C18 HPLC column (250.times.21.2 mm;)
was equilibrated in Buffer A (0.05% trifluroacetic acid ›TFA!/95% H.sub.2
O), and the HPLC system was programmed to deliver a linearly increasing
gradient of Buffer B (100% methanol) over 30 min (Buffer B=0% at 0 min;
100% at 30 min), at a flow rate of 8 ml/min. Column eluant was monitored
with an ultraviolet detector set at 254 nm, and the new peak appearing at
33 min was isolated, lyophilized and analyzed by NMR and mass
spectrometry, giving the following results, which are consistent with the
structure shown in SCHEME II and labeled as CBZ-lysine-AP.
.sup.1 H-NMR (270 MHZ, D.sub.2 O): .delta.1.32 (m, 2H), 1.62 (m, 4H), 2.99
(t, 2H, J=7.6 Hz), 3.19 (s, 2H), 3.62-3.93 (m, 6H), 5.00 (d, 1H, J=12.6
Hz), 5.08 (d, 1H, J=12.5 Hz), 7.36 (m, 5H); .sup.13 C-NMR (67.5 MHZ,
D.sub.2 O) .delta.22.2, 24.8, 31.2, 48.3, 52.8, 56.0, 64.0, 67.0, 69.0,
69.3, 69.6, 95.5, 127.7, 128.2, 128.4, 136.6, 158.0, 179.6.
Incubation of CBZ-lysine-AP with acetaldehvde
A solution of CBZ-lysine-AP (5 mg) in 2 ml of 0.2M phosphate buffer was
subjected to prolonged incubation with 10 equivalents of acetaldehyde
(about 25.degree. C.). A parallel incubation was carried out at the same
time, identical but for the absence of acetaldehyde. After five weeks,
aliquots of each incubation mixture were analyzed by the following
methods: visual inspection, AGE-ELISA (monoclonal and polyclonal
antibody-based); absorption/fluorescence spectroscopy for
excitation/emission maxima; HPLC; LC-MS (liquid chromatography-mass
spectroscopy); and .sup.1 H-NMR (proton nuclear magnetic resonance
spectroscopy).
Upon initial inspection, applicants noted that the incubation mixture which
included acetaldehyde was clear and unpigmented while the solution of
CBZ-lysine-AP incubated for five weeks in the absence of acetaldehyde was
markedly yellow in color. Applicants took this as evidence that
CBZ-lysine-AP went on, over time, to form more advanced (and pigmented)
advanced glycation products but that the formation of such yellow
pigmented AGEs was blocked by the formation of the expected
Amadori-acetaldehyde adduct (AAA) when acetaldehyde was present in the
incubation mixture. This conclusion was borne out by experimental
measurement of AGE formation.
Aliquots from the two incubations were analyzed by two distinct competitive
ELISA procedures, one based on polyclonal anti-AGE antibodies (FIGS. 1A
and and the other on a monoclonal anti-AGE antibody (FIG. 2). Both
procedures indicated that significantly more immunoreactive AGE epitopes
had developed over time in the incubation of CBZ-lysine-AP without
acetaldehyde than under identical incubation conditions but in the
presence of acetaldehyde.
Fluorescence spectrometric analysis of these two incubation mixtures
revealed the same wavelength of maximum excitation at 320 nm. The
incubation mixture of CBZ-lysine-AP incubated with acetaldehyde showed
maximal .delta..sub.em at 388 nm while the parallel incubation mixture
without acetaldehyde showed maximal .delta..sub.em at 420 nm, the latter
value consistent with the typical excitation/emission profile of AGEs.
Analysis of the incubation mixtures by HPLC (Primesphere 5.mu. C18
›250.times.21.2 mm! column; 0-50% Buffer B over 50 min at 7 ml/ min; UV
monitoring at 254 nm) revealed two major peaks present in the incubation
made with acetaldehyde that were not present in comparable HPLC analysis
of the starting material (see FIG. 3, arrows marked 1 & 2). Eluant
fractions corresponding to the central area of Peak 1 (identified in FIG.
3) were pooled, lyophilized and characterized by .sup.1 H-NMR and mass
spectrometry.
The .sup.1 H-NMR spectrum of this fraction (FIG. 4) showed the appearance
of two sets of methyl groups at .delta.1.1 and 1.27 ppm, each appearing as
a doublet attributable to vicinal coupling with the methine proton
(OCHN--). The presence of two such doublets is consistent with the
presence of diastereomeric pairs of either the 5- or the 6-membered ring
structures shown in Scheme II; or the formation of only a single
sterioisomer of the 5-membered spirobicyclic AAA and a single sterioisomer
of the 6-membered fused bicyclic AAA. The mass spectrum (FIG. 5) of this
peak displayed a single molecular ion at m/z 469 (MH+), consistent with
the identical molecular weights of the proposed structures whether they
cyclize to form a 5- or 6-membered ring AAA. Both structures may exist as
an equilibrium mixture. Other, minor products in the above reaction
mixture could be assigned preliminary compositions on the basis of mass
spectrometric data, indicating the integration of two acetaldehyde
molecules with a single CBZ-lysine-AP molecule (MS m/z 513 ›AP+2CH.sub.3
CO--H.sub.2 O!).
EXAMPLE 2
Applicants designed a second test system to define the activity of the
aldehyde agents of the present invention as inhibitors of advanced
glycation. In this in vitro system, a test protein is incubated with a
reducing sugar, optionally with mild heating to accelerate the initial
stages of the Maillard reaction. Aliquots of this pre-reacted protein
sample are then incubated either in the presence or absence of an added
aldehyde agent of the present invention, in order to measure the
inhibitory effect of such added aldehyde agent on the glycation of a
typical protein molecule under experimental browning conditions similar to
the conditions of protein aging in vivo. In a working example, BSA (50
mg/ml)is incubated in 0.1M glucose at pH 7.5 for 48 hours at 37.degree. C.
in an aqueous buffer. During this abbreviated pre-incubation period, many
of the lysine .epsilon.-amino groups of the protein condense with glucose
to form initial Schiff-base (SB) adducts, which rearrange to generate the
Amadori product (AP). After 48 hours, the BSA sample is dialyzed against
buffer to remove unreacted glucose and other low molecular weight
reactants. The protein sample then is divided into three portions (A, B,
and C) to study the kinetics of protein aging and to measure the
inhibition of this non-enzymatic glycation by typical model aldehyde
agents of the present invention. Part A is not treated with added
aldehyde, while parts B and C are incubated with an active aldehyde agent
of the present invention, in this case acetaldehyde and
4-hydroxybenzalaldehyde, respectively. Each of the experimental
preparations are incubated for five weeks at room temperature, and samples
of are were collected every week and stored at -20.degree. C. for later
analysis. At the end of the incubation, the collected samples are assessed
for AGE content by: (1) visual inspection for colored products; (2)
fluorescence spectroscopy to determine excitation/emission profiles and
wavelength maxima; and (3), AGE-ELISA (both monoclonal and polyclonal
antibody-based). By each of these tests, significantly fewer AGEs are
present in the incubations that included an added aldehyde agent of the
present invention than in control incubations without the added aldehyde
agent.
EXAMPLE 3
In vivo studies
When ethanol is consumed it is converted into acetaldehyde by the activity
of constitutive and inducible alcohol dehydrogenase enzymes. Applicants
considered that the elevation of acetaldehyde levels in vivo by chronic
consumption of ethanol would be reflected in an inhibition of the normal
accumulation of AGEs and AGE-modified proteins in the body. Because AGE
levels in normal animals, including humans, is low and AGE accumulation
progresses only slowly with aging, Applicants considered that an in vivo
demonstration of the AGE-inhibiting activity of the metabolic products of
ingested ethanol might conveniently be demonstrated in diabetic animals,
which much more rapidly accumulate AGEs and show elevated circulating and
tissue AGE levels as a consequence of their chronic hyperglycemia.
Applicants predicted that, in a diabetic population, ethanol consumption
would be clearly reflected in a diminishment of the
hyperglycemia-dependent elevation in AGE levels, because consumed ethanol
would be converted to acetaldehyde in vivo, and the consequent reaction of
this acetaldehyde with Amadori products would lock these glycation
intermediates into an unreactive closed configuration (i.e, AAA-type
adducts), thus inhibiting progression of the Maillard reaction and
lowering the accumulation of AGEs that would otherwise be expected to
occur in conjunction with diabetes. Since hemoglobin is well-known to
become spontaneously glycated, forming both hemoglobin A.sub.1c
(HbA.sub.1c ; which is modified by an Amadori product), as well as
hemoglobin-AGE (Hb-AGE; which is modified by advanced glycation end
products), Applicants considered hemoglobin to represent a convenient
substrate by which to measure the effects of alcohol consumption on in
vivo AGE levels. Applicants also recognized that measurements of the
glycation status of hemoglobin, or of other conveniently measured
substrates for AGE formation, would provide a ready marker for the recent
history of alcohol consumption by a subject.
To verify these related predictions in an animal model system, Applicants
chose to induce diabetes and hyperglycemia in rats, which could then be
fed an ethanol-supplemented diet. Later, blood samples could be tested for
Hb-AGE levels relative to controls (not fed ethanol) as an index of
alcohol consumption. Accordingly, male outbred Wistar rats (HSD:Wi),
weighing between 150-175 g (approximately 6 weeks old) were group housed
(three per cage) and provided standard laboratory rat chow and water ad
libitum during an adaptation period of one week. Half of these rats were
then treated with streptozotocin (65 mg/kg) to induce diabetes mellitus
and hyperglycemia. Diabetes was confirmed a 1 week by measurement of blood
glucose (440 +/- 38 mg/dl for the streptozotocin-treated group of rats).
Diabetic streptozotocin-treated rats were paired by matching blood glucose
levels. One rat from each pair was assigned to Group 1 and one to Group 2,
so that blood glucose levels were matched between these two groups.
Control rats (not treated with streptozotocin) were randomly assigned to
Groups 3 and 4 (non-diabetic). All animals were then placed on one of two
calorically matched liquid diets ad libitum (Bio-Serve Ethanol Diet or
Bio-Serve Caloric Control Diet; Bio-Serve, Frenchtown, N.J.). Groups I and
3 received control diet, while Groups 2 and 4 received the
ethanol-supplemented diet (36% of calories as ethanol). Pilot experiments
revealed that diabetic rats in particular also required supplemental water
under this protocol. In subsequent experiments, all animals were provided
the control liquid diet rather than standard chow during the acclimation
period, and groups scheduled to receive ethanol were switched to the
ethanol-supplemented diet according to the protocol outlined. Blood
samples were taken periodically from rats in all groups for determination
of total hemoglobin, HbA.sub.1c, Hb-AGE, and blood glucose levels.
Hb-AGE values were determined from blood samples taken two weeks after the
experimental groups were switched to ethanol-supplemented diets. Table I
below shows that Hb-AGE levels in diabetic rats fed a high ethanol diet
were significantly lower than Hb-AGE levels in diabetic rats fed an
isocaloric control diet without ethanol at the two-week point. FIG. 6
shows that Hb-AGE levels were significantly lower in diabetic rats fed a
high alcohol diet than in diabetic rats fed the iocaloric, control diet
(without alcohol) as determined from blood samples taken at approximately
one month after the initiation of the diet protocol.
As predicted HbA.sub.1c levels did not differ between the two groups,
indicating that alcohol treatment did not affect the formation of the
Amadori product, the Hb-AGE precursor. Since the assay for A.sub.1c does
not discriminate betwwen acetaldehyde-A.sub.1c and A.sub.1c, this is to be
expected. The HbA.sub.1c data also served to confirm that both
experimental groups experienced the same degree of hyperglycemia.
By way of explanation, but without limitation, this result may reflect the
covalent modification of hemoglobin-Amadori product intermediates by
acetaldehyde arising by conversion from dietary ethanol. These
acetaldehyde-reacted Amadori products will be locked in their closed
conformation, and thus inhibited from progressing through the Maillard
reaction to generate more advanced glycation products. Irrespective of the
exact chemical mechanism, ethanol consumption will be reflected
quantitatively and qualitatively in the modification of proteins by AGEs
in vivo, and measurement of the type and/or degree of such alterations in
the normal Maillard processes and products will reflect the recent history
of alcohol consumption by the subject. Note that although no significant
difference in the overall degree of advanced glycation of hemoglobin could
be detected between non-diabetic rats offered a high-ethanol diet versus a
no-ethanol diet, specific differences in the composition of glycation
products and/or intermediates may occur between these non-diabetic groups.
Although the data obtained in diabetic animals confirm, in an in vivo
situation, the inhibitory effect of ethanol on AGE formation, only a small
and not statistically significant effect was observed in the alcohol
versus non-alcohol treated non-diabetic rats. In general, non-diabetic
animals show a much lower level of protein glycation, as measured by
currently available assays. More pronounced decreases in AGEs may be
observed with longer-term ethanol administration and/or by studying
protein and tissue components that have a longer life in vivo than red
cell hemoglobin.
Specific Amadori-acetaldehyde adducts (AAAs) may occur on proteins or other
biomolecules in ethanol-fed subjects, for instance, which AAAs could be
identified by specific chemical or immunological characteristics.
TABLE I
______________________________________
Hb-AGE
Blood Glucose
Diet (mean .+-. sd)
______________________________________
Group 1 diabetic control 3.1 .+-. 0.7
Group 2 diabetic ethanol 1.5 .+-. 0.6
Group 3 normal control 1.5 .+-. 0.5
Group 4 normal ethanol 1.6 .+-. 0.4
______________________________________
EXAMPLE 4
In an attempt to identify chemically the hemoglobin A.sub.1c -acetaldehyde
adduct (HbA.sub.1c -AA) from alcohol-fed diabetic rats, a HPLC-based
method was developed to study changes to hemoglobin induced by
acetaldehyde in vivo. The hemolysates were obtained after standard workup
of the red blood cells and analyzed by a Poly CAT A cation-exchange column
in stepwise salt and Ph gradient. The mobile phases used were solution A:
40 mM Bis-Tris, 4 mM KCN, and 5 mM EDTA (pH 6.8), and solution B
consisting of solution A and 0.2M NaCl (pH 6.8). The gradient program
consisted of initial conditions of 78% A and 22% B, increasing to 56% B at
16 minutes, and 100% B at 22 minutes, and back to 22% B at 35 minutes. The
flow rate was held constant at 1.0 ml/minute and the effluent absorbency
was monitored at .lambda.415 nm. The HPLC profiles of the hemolysates
obtained from four group of rats are displayed in panels (a-d) of FIG. 7.
This method distinguished 23 hemoglobin fractions. In a comparison between
the hemolysate profile of non-diabetic and diabetic, a clear difference in
the HbA.sub.1c concentration as well as in the shape and integration of
the area eluted between 18 and 23 minutes (FIG. 7, panels a and b). Of
note, in Panel c, the HPLC profile of diabetic rats which had consumed
alcohol is similar to that of non-diabetics. In the 18-23 minute elution
range, the hemoglobin-HPLC profile of diabetic rats was restored to the
shape of non-diabetics (compare panels b and c) by alcohol consumption,
which could be due to the fixation of HbA.sub.1c by acetaldehyde.
An analysis of the globin chains from certain hemoglobin fractions from the
diabetic, ethanol diet group (above) was carried out using a reverse phase
liquid chromatography coupled to electrospray ionization mass spectrometry
(LC-MS) as shown in FIG. 8. The .alpha.- and .beta.-chains of the native,
unaltered fraction (Hb.sub.0) were eluted at about 31 and 34 minutes,
respectively (FIG. 8). The assignment of the chains was based on the
molecular weight as indicated by mass spectra of .alpha.-chain (15198
Daltons) and .beta.-chain (15861 Daltons). To determine the accuracy of
this procedure, HbA.sub.1c was also analyzed, and the results are shown in
FIG. 8.
The .alpha.- and .beta.-chains eluted under similar conditions at about 31
and 34 minutes, respectively, and the mass spectra displayed a molecular
ions of m/z 15199 (.alpha.-chain) and 16011 (.beta.-chain). As expected,
the .beta.-chain displayed an increase of 162 Daltons compared to the
parent .beta.-chain obtained from Hbo due to the glycation of the
N-terminal. Applicants next identified and characterized molecular species
from a fraction containing an HbA.sub.1c -acetaldehyde adduct similar to
our in vitro model. We expected this adduct to elute faster from the poly
CAT A column because the order of elution from cationic exchangers
correlates with isoelectric points. Therefor several faster running
fractions were analyzed by LC-MS. As expected, the LC-MS profile of one of
these fractions showed a peak at about 31.9 minutes, indicating the
presence of unmodified .alpha.-chain, as well as three other peaks which
were related to .beta.-chain based on their elution time (approximately
33.5, 34.2 and 37.7 minutes). ESMS spectra of two of these peaks displayed
a molecular ion of 16070 and 16317 Daltons and remain to be further
characterized. Interestingly enough, of note, the ESMS spectrum of the
third peak gave a molecular weight of 16040.0 Daltons (FIG. 9A) which is
consistent with the mass of the .beta.-chain of the HbA.sub.1c
+acetaldehyde-H.sub.2 O.
These studies demonstrate an efficient way to stabilize the early states of
advanced glycation by fixation of the cyclic form of the AP in vitro. Upon
stabilization of the AP with acetaldehyde, its progress towards AGEs
formation has been inhibited dramatically as indicated by AGE-ELISA
(monoclonal and polyclonal antibodies) and fluorescence spectroscopy.
Furthermore, the structure of AAA was characterized by mass spectroscopy
and .sup.1 HNMR. In a second model system in vitro, bovine serum albumin
was used as the target for advanced glycation and the inhibition of AGEs
after in vitro glycation and further treatment with acetaldehyde was
studied. After establishing the in vitro models, AGEs formation in a group
of diabetic and non-diabetic rats fed either a control or an
ethanol-supplemented diet was studied. The AGEs levels in diabetic rats
fed an ethanol diet were reduced to the range of AGEs in normal rats.
Interestingly, the HPLC profile cation exchange chromatography profile of
the red-cell hemolysate from diabetic rats on an ethanol-supplemented diet
is similar to that of normal rats. Ethanol is converted into acetaldehyde
by alcohol dehydrogenase. The dramatic decrease in the level of AGEs in
diabetic rats on an ethanol-supplemented diet may be ascribed to the
action of acetaldehyde on Amadori and Heyns rearrangement products. As
shown, acetaldehyde can target HbA.sub.1c and form a stable AAA-type
compound.
Globin chains from the diabetic, ethanol-supplemented diet group were
separated by reversed phase HPLC and analyzed by electrospray mass
spectrometry. Hb-A.sub.1c -acetaldehyde adduct was isolated in a fraction
which eluted faster than Hb-A.sub.1c in the cation exchange chromatography
and the .beta.-chain showed an apppropiate molecular weight increase due
to the attachment of acetaldehyde to the early glycation product,
HbA.sub.1c.
EXAMPLE 5
A variety of aldehydes are expected to be active in inhibiting the
undesired browning of proteins and other biomolecules, either in vitro or
in vivo or both. Assays are provided for the discovery of such activity,
so that potentially useful inhibitors of browning may be discovered. A
sample of soluble protein (BSA, for instance), which is known to be
subject to advanced glycation, may be incubated in the presence of glucose
or another reducing sugar, in aqueous buffer at neutral pH, for example,
such that the protein will accumulate modifications by advanced glycation.
If various aldehyde test agents then are separately included in parallel
incubations, those aldehydes that are active in inhibiting the Maillard
reaction may conveniently be identified by the lower amount of AGEs that
accumulate on the sample protein in the presence of a test aldehyde
compared to the number of AGEs that accumulate in the absence of such an
aldehyde agent. For convenience, AGEs may be identified by any of several
methods well known in the art, e.g., visual inspection for colored
products, tests for protein crosslinking, fluorescence spectrophotometric
tests for characteristic absorbance/emission maxima, AGE receptor-based
assays, and AGE-specific immunoassays including, for instance,
AGE-specific ELISAs based on anti-AGE antibodies. Optionally, the test
incubations may be sealed, and the temperature of the incubation
artificially elevated (as by a constant temperature bath) to accelerate
the Maillard processes so that browned products accumulate more quickly.
The addition of chelating agents and antioxidants may optionally be
included so as to reduce the number of confounding metal-catalyzed and
oxidizing reactions in the incubation mixture. Dose/response protocols, in
which the aldehyde test agents are present in various doses are useful to
identify potential inhibitors of advanced glycation of different
potencies; those aldehydes which are active at lower concentrations would
constitute a preferred group to further examine for additionally desirable
characteristics, depending on the specific contemplated use. In accordance
with this assay method, for instance, the various GRAS (i.e., Generally
Recognized As Safe and so listed by the U.S. FDA) aldehydes can be tested
for efficacy in inhibiting advanced glycation. Particularly effective GRAS
compounds could then be further tested for compatibility as agents useful
as Maillard inhibitors in vivo (e.g., to prevent protein aging or diabetic
complications) or in vitro (e.g., to prevent food spoilage, the
development of unpalatability, or inactivation of chemical or
pharmaceutical compounds caused by non-enzymatic glycation and
crosslinking during prolonged storage). Presently used food preservatives
and discoloration preventives such as sulfur dioxide, known to cause
toxicity including allergy and asthma in animals, might be replaced with
compounds such as those described herein; such alternative inhibitors of
Maillard reactions in foodstuffs and other comestibles are highly
desirable. Agents of the present invention are of utility in this regard,
as well as in pharmaceutical compositions to inhibit advanced glycation in
vivo. Use in vivo is particularly indicated in aging and diabetes when the
accumulation of AGEs in the body causes a number of pathogenic processes,
both directly due to the chemical reactivity of AGEs and indirectly due to
the interaction of AGEs with specialized cellular receptor systems, which
interaction triggers a wide variety of normal and pathophysiological
cellular responses.
EXAMPLE 6
Acetaldehyde-Amadori adduct (AAA) and related compounds of the present
invention also find utility as antigens or haptens, to elicit antibodies
specifically directed to AAA and AAA-like structures. Such antibodies,
likewise of the present invention, are useful in turn to identify AAA
structures of the present invention. By constructing immunoassays
employing anti-AAA antibodies of the present invention, for instance, the
degree to which proteins are modified by AAAs can be measured. As
discussed above, and depending on the half-life of the protein so
modified, immunochemical measurement of AAA epitopes on a protein sample,
such as hemoglobin, provides an index of recent alcohol (ethanol)
consumption. Likewise, immunochemical detection of AAA epitopes on
circulating and/or tissue proteins can be used to monitor the course of
therapy with agents of the present invention, which aldehyde-based agents
are directed toward inhibition of advanced glycation by locking
spontaneously formed Amadori products in their closed, and hence
unreactive, conformation.
AAA-modified BSA for use as an immunogen can be prepared according to the
following reaction Scheme III below.
##STR4##
AAA may also be synthesized ab initio by the following procedure shown in
Scheme IV below.
##STR5##
In this reaction sequence, the desired AAA of formula VII is prepared by
acetylation of the synthetic Amadori product, CBZ-lysine-AP, shown in
Scheme II, with acetic anhydride in the presence of pyridine to afford the
pentaacetylated compound. Treatment of this compound with trifluoroacetic
acid in dichloromethane affords the bicyclic system. Deacetylation of the
tetracetylated bicyclic compound, followed by NaBH.sub.4 reduction of the
intact double bond within the five-membered ring yields the desired
product.
Various haptens, antigens, and conjugated immunogens corresponding to the
aldehyde-modified Amadori products of the present invention, including
without limitation the acetaldehyde-modified CBZ-lysine-Amadori product
described in Example 1, can conveniently be prepared, either by isolation
from incubation mixtures or by direct synthetic approaches. CBZ-lysine-AP,
for example, can be incubated with acetaldehyde in vitro and isolated,
essentially as described in Example 1 (above). This AAA may then be used
as an immunogen to raise a variety of antibodies which recognize specific
eptitopes or molecular features thereof. In a preferred embodiment, AAA
itself is considered a hapten, which is correspondingly coupled to any of
several preferred carrier proteins, including for instance keyhole limpet
hemocyanin (KLH), thyroglobulin, and most preferred, bovine serium albumin
(BSA), using any of a number of well-known divalent coupling reagents such
as a carbodiimide like EDC, according to protocols widely circulated in
the art. Alternatively, the desired aldehyde-reacted Amadori product can
be synthesized ab initio, essentially as described in Scheme IV for the
Amadori-acetaldehyde adduct (AAA). Irrespective of the source, the AAA or
other aldehyde-reacted Amadori product (or Heyns product), whether alone
or coupled to a carrier protein, may be employed in any well-recognized
immunization protocol to generate antibodies and related immunological
reagents that are useful in a number of applications owing to the
specificity of the antibodies for molecular features of the
aldehyde-reacted Amadori product.
Following a preferred protocol, any of several animal species may be
immunized to produce polyclonal antisera directed against the AAA-carrier
protein conjugate, including for instance mice, rats, hamsters, goats,
rabbits, and chickens. The first of three of the aforesaid animal species
are particularly desired choices for the subsequent production of
hybridomas secreting hapten-specific monoclonal antibodies. The production
of said hybridomas from spleen cells of immunized animals may
convenientally be accomplished by any of several protocols popularly
practiced in the art, and which describe conditions suitable for
immortalization of immunized spleen cells by fusion with an appropriate
cell line, e.g. a myeloma cell line. Said protocols for producing
hybridomas also provide methods for selecting and cloning immune
splenocyte/myeloma cell hybridomas and for identifying hybridomas clones
that stably secrete antibodies directed against the desired eptiope(s).
Animal species such as rabbit and goat are more commonly employed for the
generation of polyclonal antisera, but regardless of whether polyclonal
antisera or monoclonal antibodies are desired ultimately, the
hapten-modified carrier protein typically is initally administered in
conjunction with an adjuvant such as Complete Freund's Adjuvant.
Immunizations may be administered by any of several routes, typically
intraperitoneal, intramuscular or intradermal; certain routes are
preferrred in the art according to the species to be immunized and the
type of antibody ultimately to be produced. Subsequently, booster
immunizations are generally administered in conjunction with an adjuvant
such as alum or Incomplete Freund's Adjuvant. Booster immunizations are
administered at intervals after the inital immunization; generally one
month is a suitable interval, with blood samples taken between one and two
weeks after each booster immunization. Alternatively, a variety of
so-called hyperimmunication schedules, which generally feature booster
immunizations spaced closer together in time, are sometimes employed in an
effort to produce anti-hapten antibodies preferentially over anti-carrier
protein antibodies.
The antibody titers in post-boost blood samples can be compared for
hapten-specific immune titer in any of several convenient formats
including, for instance, Ouchterlony diffusion gels and direct ELISA
protocols. In a typical direct ELISA, a defined antigen is immobilized
onto the assay well surface, typically in a 96-well or microtiter plate
format, followed by a series of incubations separated by rinses of the
assay well surface to remove unbound binding partners. By way of
non-limiting example, the wells of an assay plate may receive a dilute,
buffered aqueous solution of the hapten/carrier conjugate, preferably
wherein the carrier protein differs from that used to immunize the
antibody-producing animal to be tested; e.g. serum from AAA/KLH
conjugate-immunized animal might be tested against assays wells decorated
with immobilized AAA/BSA conjugate. Alternatively, the assay surface may
be decorated by incubation with the hapten alone. Generally, the surface
of the assay wells is then exposed to a solution of an irrelevant protein,
such as casein, to block unoccupied sites on the plastic surfaces. After
rinsing with a neutral buffered solution that typically contains salts and
a detergent to minimize non-specific interactions, the well is then
contacted with one of a serial dilution of the serum prepared from the
blood sample of interest (the primary antiserum). After rinsing again, the
extent of test antibodies immobilized onto the assay wells by interaction
with the desired hapten or hapten/carrier conjugate can be estimated by
incubation with a commercially available enzyme-antibody conjugate,
wherein the antibody portion of this secondary conjugate is directed
against the species used to produce the primary antiserum; e.g. if the
primary antiserum was raised in rabbits, a commercial preparation of
anti-rabbit antibodies raised in goat and conjugated to one of several
enzymes, such as horseradish peroxidase, can be used as the secondary
antibody. Following procedures specified by the manufacturer, the amount
of this secondary antibody can then be estimated quantitativley by the
activity of the assoicated conjugate enzyme in a calorimetric assay. Many
related ELISA or radioimmunometric protocols, such as competitive ELISAs
or sandwich ELISAs, all of which are well-known in the art, may optionally
be substituted, to identify the desired antisera of high titer; that is,
the particular antisera which give a true positive result at high dilution
(e.g. greater than 1/1000 and more preferably greater than 1/10,000).
Similar immunometric protocols can be used to estimate the titer of
antibodies in culture supernatants from hybridomas prepared from spleen
cells of immunized animals. In so characterizing antisera or hybridoma
supernatants, it is desirable to employ a variety of control incubations.
e.g. with different carrier proteins, related but structually distinct
haptens or antigens, and omitting various reagents in the immunometric
procedure in order to minimize non-specific signals in the assay and to
identify reliable determinations of antibody specificity and titer from
false positive and false negative results. The types of control
incubations to use in this regard are well known. Also, the same general
immunometric protocols subsequently may be employed with the antisera
identified by the above procedures to be of high titer and to be directed
against specific structural determinants in the aldehyde-modified Amadori
products on biological samples, foodstuffs or other comestibles, or other
amine-bearing substances and biomolecules of interest. Such latter
applications of the desired anti-aldehyde-modified Amadori product
antibodies, whether polyclonal or monoclonal, together with instructions
and optionally with other useful reagents and diluents, including, without
limitation, a set of molecular standards of the aldehyde-modified Amadori
product, may be provided in kit form for the convience of the operator.
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